It has previously been proposed to employ enzyme electrodes having laminated membranes for assaying glucose as described for example in Clark U.S. Pat. No. 3,539,455; Newman U.S. Pat. Nos. 3,979,274 and 4,073,713; Johnson U.S. Pat. Nos. 4,220,503, 4,356,074 and 4,404,066; and Japanese Patent Application publication No. 60-185153. Such enzyme electrode assays of glucose involve measurement of the enzyme-catalyzed oxidation of glucose in accordance with the following reaction: ##STR1## The enzyme, glucose oxidase, is interposed and immobilized between two membranes, the first or outer of which comes into contact with the sample to be assayed and permits access of glucose and of oxygen to the enzyme from the sample while restricting the passage of proteins, red blood cells, and other macromolecules, and the second of which is in close relationship with the face of the sensor electrode and permits access of hydrogen peroxide to the electrode while at the same time excluding passage of interfering substances having a molecular weight greater than about 250, e.g., ascorbic acid, uric acid, and salicylic acid. In practice, the sample to be assayed, which contains both glucose and oxygen, is brought into contact with the outer face of the first or outer membrane. Diffusion of the sample through the membrane into contact with the immobilized enzyme leads to the reaction set forth above, and diffusion of the resulting hydrogen peroxide through the second or inner membrane into contact with the sensor electrode causes development of an electrical current which can then be read by conventional means, thus enabling determination of the glucose by calculations based upon similar measurements made on standard solutions containing known concentrations of glucose. The membrane-covered electrode face can be brought into contact with the sample solution either by immersing it in a bath of the sample or by providing a flow cell or chamber through which the sample is passed across the outer face of the first or outer membrane. However, glucose assays conducted as in the prior art have lacked accuracy in the case of solutions having high concentrations of glucose, as are often found for example in undiluted whole blood or serum. It has consequently been the practice to dilute such high-concentration samples by a suitable amount of buffer before assay. The dilution step is time-consuming and is itself an additional source of possible error.
It has now been found that the thickness and pore size of the first or outer membrane in contact with the sample is critical for achieving consistent and accurate measurements, particularly when the samples to be assayed have a high concentration of glucose. The membranes of the prior art have generally been so highly permeable to the passage of glucose that, particularly in the case of samples having high concentrations of glucose, the amount of glucose coming into contact with the immobilized enzyme exceeds the amount of oxygen available. Consequently, the oxygen concentration is the rate-limiting component of the reaction rather than the glucose concentration, so that the accuracy of the glucose assay is destroyed. Equally critical is the thickness and pore size of the second or inner membrane, which must be sufficiently permeable to permit passage of the hydrogen peroxide to the electrode surface as rapidly as it is formed, but which should not permit the ready passage of potential interfering substances. Retention of hydrogen peroxide by this membrane, that is, failure to permit rapid passage of hydrogen peroxide from the immobilized enzyme to the electrode face can upset the equilibrium of the reaction and lead to erroneous results; it also can lower sample throughout and increase reagent usage.
The present invention by employing an electrode assembly having a first or outer membrane having a thickness of 1 to 20 .mu.m, preferably 5 to 7 .mu.m, and a pore size of 10 to 125 .ANG., which limits the diffusion of glucose molecules through the membrane, ensures the presence of sufficient oxygen in contact with the immobilized enzyme. Moreover, by providing a second or inner membrane having a thickness of 2 to 4 .mu.m, more preferably 2 to 3 .mu.m, the present invention provides sufficient permeability to ensure rapid removal of hydrogen peroxide from the enzyme into contact with the sensor electrode and rapid achievement of an equilibrium state.
In using the electrode of the present invention for assaying glucose, the electrode may be maintained in contact with the sample until the reaction attains equilibrium, after which an amperometric measurement is taken and compared with that of a standard solution taken under the same conditions, a procedure which requires of the order of 10 to 30 seconds. Additional time is required to wash residual sample from the outer face of the first membrane and allow the sensor electrode current to return to its base line value, so that in the case of an electrode used for successive assays of different samples, the total time for each sample is of the order of 60-80 seconds, depending upon the concentration of glucose.
In using the electrode of the present invention, in the conventional procedure of waiting for equilibrium to be established before measuring electrode current, the time required for assaying a succession of different samples is somewhat less than the time required using electrodes of the prior art. However, the electrode of the present invention has been found to make it possible to employ a different and much more rapid procedure, particularly beneficial in the case of samples having high glucose concentrations. It has been found that accurately reproducible assays can be achieved with the electrode of the present invention by making the amperometric measurement before reaction equilibrium has been achieved. Instead of waiting for equilibrium to be attained, the measurement is made at an arbitrary and standard time while the reaction in the cell is still approaching equilibrium. Preferably, the time of measurement is not less than 5 seconds after the sample is first brought into contact with the first or outer membrane. More preferably, to obtain the most reproducible results the time of measurement should be selected such that the current has achieved at least 90% of the steady state value. Contact of the sample with the membrane need not be thereafter continued, and preferably the sample is displaced as rapidly as possible by water or a buffer solution in order to return the electrode current to its base line value. This allows for a high throughput that is particularly advantageous when the analyzer containing the sensor has other sensors as well. Moreover, making the reading at such an early time has the advantage of limiting the effects of interfering substances, which generally take longer to move through the inner membrane than H.sub.2 O.sub.2 ; thus, a good H.sub.2 O.sub.2 reading is obtained with limited interference from the substances, which have yet to get through the membrane.
Alternatively, the electrode and its laminated membrane can be combined with a flow-through sample chamber or cell of limited size for presenting the sample to the face of the first membrane; the volume or size of the sample and the rate of flow can be so selected as to provide a standard short time of residence of the sample in contact with the outer membrane. In this embodiment, the sample is preferably of a fixed standard volume or size and is forced through the chamber across the face of the membrane via a peristaltic pump. Consequently, only a minimal quantity of sample, in the form of a thin surface film, remains in the chamber after passage of the specimen. This residual film of sample is rapidly consumed and can be rapidly removed by flowing through the chamber a small quantity of buffer, thus restoring the current to the base line value and readying the sensor for assay of a new sample.